Fast and integrated fluorescence lifetime microscopy

• Subject Area: Cell Biology
• Term: since 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 413956898

We apply for a fast and highly integrated Fluorescence Lifetime Microscope (FLIM) with single and multi-photon excitation.Fluorescent labels for microscopic structures of interest are universally used in the biomedical sciences. Most applications make use only of the spectral properties of fluorochromes, their color. They do have another property which can be exploited, the duration of the time period between excitation and emission of a photon, their lifetime. This lifetime differs from fluorochrome to fluorochrome and thus can be used for discrimination in addition to the spectral response. Furthermore, the lifetime of certain fluorochromes changes depending on local environmental conditions. FLIM thus can reveal information about molecular interactions and the metabolic or signaling state within the cell. However, FLIM used to be slow and difficult to perform so that it was applied only in few niches of biomedical research by dedicated specialists.Recent technological advances now allow much higher recording speeds than previously possible. The new system is two orders of magnitudes faster. Images that used to be recorded in minutes can now be acquired in seconds or less, at the typical speed of a confocal microscope. In addition, the new technology is much easier to apply and more versatile. It is fully integrated in the normal microscope software with a user-friendly interface. Therefore, while previous setups required a specialist for operation the new system can be operated by all life science microscopists with confocal experience. The scientific projects described below prove that these advances allow new experimental designs, for example in life cell and live animal (intravital) microscopy for which previous FLIM setups were too slow. Even in non-moving objects like tissue sections or plants, the significantly shorter recording times allow a respectively higher throughput of samples. What used to be a niche application due to technical limitations can now be broadly applied to a large number of research fields. The DFG initiative provides the opportunity to strongly accelerate the availability of this game changing technological development to the research community. With our application we plan to establish this new technology at the heart of physiological research, with neurobiological and immunological applications, complemented by applications in plants and method development. Published studies successfully applied FLIM in three areas: separation of spectrally overlapping fluorochromes, lifetime changes of applied or endogenous sensor molecules (e.g. Ca2+, NAD(P)H), and lifetime changes due to Förster Resonance Energy Transfer (FRET). The projects described below comprise examples for all three application areas with approaches which only now become broadly applicable thanks to the now achievable speed of the technique.

• DFG Programme: Major Instrumentation Initiatives
• Major Instrumentation: Fluorescence Lifetime Microscope (FLIM) with single and multi-photon excitation
• Instrumentation Group: 5090 Spezialmikroskope
• Applicant Institution: Ludwig-Maximilians-Universität München
• Leader: Privatdozent Dr. Steffen Dietzel
• Participating Persons: Professorin Dr. Antje Grosche; Professorin Dr. Magdalena Götz; Professor Dr. Martin Kerschensteiner; Professor Dr. Don C. Lamb; Professor Dr. Steffen Massberg; Professor Dr. Jovica Ninkovic; Professor Dr. Martin Parniske; Professorin Barbara Schraml-Schotta, Ph.D.; Professor Dr. Markus Sperandio; Professorin Dr. Barbara Walzog